Motor neuron circuits control respiration, coordinated movement and sympathetic nervous system activity in vertebrates. A promising treatment for motor neuron degenerative diseases is using cell therapies to replace lost motor neurons. The first step in developing such a treatment will be to create a strategy for directing replacement neurons to form functional motor neuron circuits. During embryonic development, motor neuron circuits are formed through the generation of multiple motor neuron subtypes, which each project to specific muscle targets. Differential gene expression distinguishes distinct motor neuron subtypes and contributes to the differential axon pathfinding of these neurons. It is extremely difficult to isolate individual motor neuron subtypes from intact spinal cords. Because of this challenge, there is relatively little known about the genetic mechanisms that underlie motor neuron subtype specification and axon guidance. The goal of this proposal is to identify novel genes that are differentially expressed in different motor neuron subtypes, and to investigate their functions in the context of motor neuron circuit formation. Preliminary results using a novel technique called 4-Thiouracil (4-TU) tagging, combined with high-throughput RNA-sequencing (RNA-seq), have revealed numerous genes that are enriched in brachial motor neurons compared to thoracic motor neurons. 4-TU tagging uses the cell-type-specific expression of the enzyme Uracil Phosphoribosyl Transferase (UPRT) to label newly synthesized RNAs in specific subsets of cells in the developing spinal cord. Because the brachial spinal cord contains Lateral Motor Column (LMC) neurons and the thoracic spinal cord does not, genes that are enriched in the brachial spinal cord are likely enriched in LMC neurons. Aim1 will investigate LMC specification and axon guidance by first examining the expression of 14 brachial motor neuron genes. From these, I will use genes that are confirmed to be specifically expressed in LMC neurons for gain of function and loss of function experiments. LMC specification will be examined by immunostaining for LMC-specific markers. LMC axon pathfinding will be assessed by labeling motor axons with GFP and through retrograde labeling experiments. Aim 2 will identify novel genes that are differentially expressed in medial LMC (LMCm) neurons and Medial Motor Column (MMCm) neurons, by performing 4-TU tagging and RNA-seq experiments in these two motor neuron subtypes. The expression of 5 putative LMCm and 5 putative MMCm genes will be examined through co-immunostaining for motor neuron subtype specific genes. LCMm and MMCm specific genes will then be used for gain of function and loss of function experiments. Motor neuron subtype specification and axon pathfinding will be assessed by immunostaining for LMCm or MMCm specific markers, labeling motor neuron axons, and through retrograde labeling. These experiments will be the first unbiased examination of motor neuron subtype specific gene expression in developing embryos.